Thonzonium bromide inhibits progression of malignant pleural mesothelioma through regulation of ERK1/2 and p38 pathways and mitochondrial uncoupling.

BACKGROUND: Malignant Pleural Mesothelioma (MPM) is a rare malignancy with a poor prognosis. Current therapies are unsatisfactory and novel cures are urgently needed. In a previous drug screening, we identified thonzonium bromide (TB) as one of the most active compounds against MPM cells. Since the biological effects of TB are poorly known, in this work we departed from some hints of previous studies and investigated several hypotheses. Moreover, we evaluated the efficacy of TB in an in vivo xenograft rodent model. METHODS: In vitro assessment was made on five MPM (Mero-14, Mero-25, Ren, NCI-H28, MSTO-211H) and one SV40-immortalized mesothelial cell line (MeT-5A). We evaluated TB ability to affect proliferation, apoptosis, mitochondrial functions and metabolism, and the mevalonate pathway. In vivo assay was carried out on MPM-xenograft NOD-SCID mice (4 mg/kg delivered intraperitoneally, twice a week for 4 weeks) and the overall survival was analysed with Kaplan-Meier curves. RESULTS: After TB treatment, we observed the suppression of ERK 1/2 phosphorylation, the increase of BAX expression and p38 phosphorylation. TB affected Ca2+ homeostasis in both mitochondrial and cytosolic compartments, it regulated the mitochondrial functioning, respiration, and ATP production as well as the mevalonate pathway. The in vivo study showed an increased overall survival for TB treated group vs. vehicle control group (P = 0.0076). CONCLUSIONS: Both in vitro and in vivo results confirmed the effect of TB on MPM and unravelled novel targets with translational potential.

Drug-repositioning screening identified fludarabine and risedronic acid as potential therapeutic compounds for malignant pleural mesothelioma.

Objectives Malignant pleural mesothelioma (MPM) is an occupational disease mainly due to asbestos exposure. Effective therapies for MPM are lacking, making this tumour type a fatal disease. Materials and Methods In order to meet this need and in view of a future "drug repositioning" approach, here we screened five MPM (Mero-14, Mero-25, IST-Mes2, NCI-H28 and MSTO-211H) and one SV40-immortalized mesothelial cell line (MeT-5A) as a non-malignant model, with a library of 1170 FDA-approved drugs. Results Among several potential compounds, we found that fludarabine (F-araA) and, to a lesser extent, risedronic acid (RIS) were cytotoxic in MPM cells, in comparison to the non-malignant Met-5A cells. In particular, F-araA reduced the proliferation and the colony formation ability of the MPM malignant cells, in comparison to the non-malignant control cells, as demonstrated by proliferation and colony formation assays, in addition to measurement of the phospho-ERK/total-ERK ratio. We have shown that the response to F-araA was not dependent upon the expression of DCK and NT5E enzymes, nor upon their functional polymorphisms (rs11544786 and rs2295890, respectively). Conclusion This drug repositioning screening approach has identified that F-araA could be therapeutically active against MPM cells, in addition to other tumour types, by inhibiting STAT1 expression and nucleic acids synthesis. Further experiments are required to fully investigate this.

Tissue expression of lactate transporters (MCT1 and MCT4) and prognosis of malignant pleural mesothelioma (brief report).

BACKGROUND: Malignant pleural mesothelioma (MPM) is an aggressive neoplasm of the pleura, mainly related to asbestos exposure. As in other solid tumors, malignant cells exhibit high glucose uptake and glycolytic rates with increased lactic acid efflux into the interstitial space. Lactate transport into and out of cells, crucial to maintaining intracellular pH homeostasis and glycolysis, is carried out by monocarboxylate transporters (MCTs) and the chaperone basigin (CD147). We set out to examine the clinical significance of basigin, MCT1 and MCT4 in the context of MPM and to evaluate their expression in relation to the evolution of the disease. METHODS: We used immunohistochemistry to measure the expression of basigin, MCT1 and MCT4 in a cohort of 135 individuals with MPM compared to a series of 15 non-MPM pleura specimens. Moreover, by Kaplan-Meier and Cox analyses we evaluated whether an expression over the average of these markers could be associated with the patients' overall survival (OS). RESULTS: We detected positive staining of basigin, MCT1, and MCT4 in most MPM specimens. In particular, MCT4 was always positive in malignant tissues but undetectable in the 4 normal pleural specimens incorporated within the tissue microarray. This was confirmed in the additional series of 15 normal pleural samples. Moreover, MCT4 expression was significantly associated with reduced OS. CONCLUSION: In this study, the tissue expression of basigin did not prove to be exploitable as a diagnostic or prognostic marker for MPM patients. The expression of MCT1 was not informative either, being tightly correlated with that of basigin. However, the expression of MCT4 showed promise as a diagnostic/therapeutic and prognostic biomarker.

EIF4G1 and RAN as Possible Drivers for Malignant Pleural Mesothelioma.

For malignant pleural mesothelioma (MPM) novel therapeutic strategies are urgently needed. In a previous study, we identified 51 putative cancer genes over-expressed in MPM tissues and cell lines. Here, we deepened the study on nine of them (ASS1, EIF4G1, GALNT7, GLUT1, IGF2BP3 (IMP3), ITGA4, RAN, SOD1, and THBS2) to ascertain whether they are truly mesothelial cancer driver genes (CDGs) or genes overexpressed in an adaptive response to the tumoral progression ("passenger genes"). Through a fast siRNA-based screening, we evaluated the consequences of gene depletion on migration, proliferation, colony formation capabilities, and caspase activities of four MPM (Mero-14, Mero-25, IST-Mes2, and NCI-H28) and one SV40-immortalized mesothelial cell line (MeT-5A) as a non-malignant model. The depletion of EIF4G1 and RAN significantly reduced cell proliferation and colony formation and increased caspase activity. In particular, the findings for RAN resemble those observed for other types of cancer. Thus, we evaluated the in vitro effects of importazole (IPZ), a small molecule inhibitor of the interaction between RAN and importin-ß. We showed that IPZ could have effects similar to those observed following RAN gene silencing. We also found that primary cell lines from one out of three MPM patients were sensitive to IPZ. As EIF4G1 and RAN deserve further investigation with additional in vitro and in vivo studies, they emerged as promising CDGs, suggesting that their upregulation could play a role in mesothelial tumorigenesis and aggressiveness. Furthermore, present data propose the molecular pathways dependent on RAN as a putative pharmacological target for MPM patients in the view of a future personalized medicine.

Mesothelin promoter variants are associated with increased soluble mesothelin-related peptide levels in asbestos-exposed individuals.

BACKGROUND: Soluble mesothelin-related peptide (SMRP) is a promising diagnostic biomarker for malignant pleural mesothelioma (MPM), but various confounders hinder its usefulness in surveillance programmes. We previously showed that a single nucleotide polymorphism (SNP) within the 3'untranslated region (3'UTR) of the mesothelin (MSLN) gene could affect the levels of SMRP. OBJECTIVES: To focus on SNPs located within MSLN promoter as possible critical genetic variables in determining SMRP levels. METHODS: The association between SMRP and SNPs was tested in 689 non-MPM subjects and 70 patients with MPM. Reporter plasmids carrying the four most common haplotypes were compared in a dual luciferase assay, and in silico analyses were performed to investigate the putative biological role of the SNPs. RESULTS: We found a strong association between serum SMRP and variant alleles of rs3764247, rs3764246 (in strong linkage disequilibrium with rs2235504) and rs2235503 in non-MPM subjects. Inclusion of the genotype information led to an increase in SMRP specificity from 79.9% to 85.5%. Although not statistically significant, the group with MPM showed the same trend of association. According to the in vitro luciferase study, rs3764247 itself had a functional role. In silico approaches showed that the binding sites for transcription factors such as Staf and ZNF143 could be affected by this SNP. The other SNPs were shown to interact with each other in a more complex way. CONCLUSIONS: These data support the suggestion that SMRP performance is affected by individual (ie, genetic) variables and that MSLN expression is influenced by SNPs located within the promoter regulatory region.

[Update of the Pdta Net Observatory of the ReS Foundation with all regional Care Pathways approved until 2023.] / Aggiornato l'Osservatorio Pdta Net di Fondazione ReS con tutti i Pdta regionali approvati fino al 2023.

Pdta Net, established and managed by Research and Health Foundation (ReS), is a database aimed at gathering and analysing the Regional Care Pathways (CPs) approved in Italy. A comprehensive search was conducted within institutional websites to retrieve all CPs approved by Italian Regions and Autonomous Provinces until December 2023, by utilizing specific keywords. Compared to the previous year, 51 new approvals were recorded. By now, Pdta Net collects 856 CPs, of which 476 are for high-impact chronic diseases and 380 for rare diseases.

Cardiovascular events after exacerbations of chronic obstructive pulmonary disease: Results from the EXAcerbations of COPD and their OutcomeS in CardioVascular diseases study in Italy.

INTRODUCTION: Exacerbations of chronic obstructive pulmonary disease (COPD) can increase the risk of severe cardiovascular events. OBJECTIVE: Assess the crude incidence rates (IR) of cardiovascular events and the impact of exacerbations on the risk of cardiovascular events within different time periods following an exacerbation. METHODS: COPD patients aged ≥45 years between 01/01/2015 and 12/31/2018 were identified from the Fondazione Ricerca e Salute administrative database. IRs of severe non-fatal and fatal cardiovascular events were obtained for post-exacerbation time periods (1-7, 8-14, 15-30, 31-180, 181-365 days). Time-dependent Cox proportional hazard models compared cardiovascular risks between periods with and without exacerbations. RESULTS: Of 216,864 COPD patients, >55 % were male, mean age was 74 years, frequent comorbidities were cardiovascular, metabolic and psychiatric. During an average 34-month follow-up, 69,620 (32 %) patients had ≥1 exacerbation and 46,214 (21 %) experienced ≥1 cardiovascular event. During follow-up, 55,470 patients died; 4,661 were in-hospital cardiovascular-related deaths. Among 10,269 patients experiencing cardiovascular events within 365 days post-exacerbation, the IR was 15.8 per 100 person-years (95 %CI 15.5-16.1). Estimated hazard ratios (HR) for the cardiovascular event risk associated with periods post-exacerbation were highest within 7 days (HR: 34.3, 95 %CI: 33.1-35.6), especially for heart failure (HR 50.6; 95 %CI 48.6-52.7) and remained elevated throughout 365 days (HR 1.1, 95 %CI 1.02-1.13). CONCLUSIONS: COPD patients in Italy are at high risk of severe cardiovascular events following exacerbations, suggesting the need to prevent exacerbations and possible subsequent cardiovascular events through early interventions and treatment optimization.

Polymorphism Pro64His within galectin-3 has functional consequences at proteome level in thyroid cells.

Introduction: The single nucleotide polymorphism (SNP) rs4644 at codon 64 of galectin-3 (gal-3, gene name: LGALS3), specifying the variant proline (P64) to histidine (H64), is known to affect the protein's functions and has been associated with the risk of several types of cancer, including differentiated thyroid carcinoma (DTC). Materials and methods: To deepen our understanding of the biological effects of this SNP, we analyzed the proteome of two isogenic cell lines (NC-P64 vs. NA-H64) derived from the immortalized non-malignant thyrocyte cell line Nthy-Ori, generated through the CRISPR-Cas9 technique to differ by rs4644 genotype. We compared the proteome of these cells to detect differentially expressed proteins and studied their proteome in relation to their transcriptome. Results: Firstly, we found, consistently with previous studies, that gal-3-H64 could be detected as a monomer, homodimer, and heterodimer composed of one cleaved and one uncleaved monomer, whereas gal-3-P64 could be found only as a monomer or uncleaved homodimer. Moreover, results indicate that rs4644 influences the expression of several proteins, predominantly upregulated in NA-H64 cells. Overall, the differential protein expression could be attributed to the altered mRNA expression, suggesting that rs4644 shapes the function of gal-3 as a transcriptional co-regulator. However, this SNP also appeared to affect post-transcriptional regulatory mechanisms for proteins whose expression was oppositely regulated compared to mRNA expression. It is conceivable that the rs4644-dependent activities of gal-3 could be ascribed to the different modalities of self-dimerization. Conclusion: Our study provided further evidence that rs4644 could affect the gal-3 functions through several routes, which could be at the base of differential susceptibility to diseases, as reported in case-control association studies.

Strategies for single base gene editing in an immortalized human cell line by CRISPR/Cas9 technology.

The use of CRISPR/Cas9 system has rapidly grown in the last years. Here, the optimization of gene editing of a single-nucleotide polymorphism in a human non-malignant somatic cell line of thyrocytes (Nthy-Ori) was described highlighting strategies for overcoming the problems concerning the delivery and off-targets. We employed both lentivirus and chemical lipids as delivery agents and two strategies for creating the double-strand breaks (DSB). The former induced a DSB by a classical Cas9 nuclease (standard strategy), while the second one employed a modified Cas9 creating a single-strand break (SSB). The knock-in was carried out using a single-stranded donor oligonucleotide or the HR410-PA donor vector (HR). The desired cells could be obtained by combining the double nickase system with the HR vector transfected chemically. This result could be due to the type of DSB, likely processed mainly by non-homologous end joining when blunt (standard strategy) and by HR when overhanging (double nickase). Our results showed that the double nickase is suitable for knocking-in the immortalized Nthy-Ori cell line, while the standard CRISPR/Cas9 system is suitable for gene knock-out creating in/del mutations. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03878-4.

[A view of care pathways approved by Italian Regions, to face the challenge of the community-based healthcare: a quali-quantitative analysis of the Pdta Net database.] / Conoscere i percorsi diagnostico-terapeutici assistenziali (Pdta) regionali approvati in Italia per affrontare il cambiamento della assistenza di prossimità: analisi quali-quantitativa del database Pdta Net.

INTRODUCTION: The adoption of a Care Pathway (CP) allows the healthcare management of patients suffering from high-epidemiological impact chronic diseases. The continuity of care of these patients is one of the main purposes of the community-based healthcare reform, foreseen in the 6th Mission of the National recovery and resilience plan. Fondazio-ne Ricerca e Salute (ReS) collects and analyses regional CPs approved in Italy, through the Pdta Net database. METHODS: Fondazione ReS has retrieved all the CPs approved by Italian Regions and Autonomous provinces until 12/31/2021 within institutional websites, through specific keywords. The quali- and quantitative analysis of CPs was based on the approving Region, the publication year, the disease (distinguishing between high-epidemiological impact chronic diseases and rare conditions) and clinical area. Following the 5-year experience gained by Fondazione ReS in terms of CPs' aims and organization for the full realization of an evidence-based healthcare of chronic patients, all data collected until 12/31/2021 underwent an in-depth double-blinded quality control. This control was aimed to make the Pdta Net database as representative as possible of the existing documents closest to a real CP. RESULTS: From 2005 to 2021, 729 regional CPs have been approved: 404 on high-impact chronic diseases and 220 on rare conditions. The CPs of chronic diseases, mostly edited by Piemonte (45 CPs), Campania (34) and Toscana (33) Regions, mainly concern on diabetes (19), chronic obstructive pulmonary disease (15), heart failure (13), stroke, multiple sclerosis and colorectal neoplasms (12 each one), breast cancer (11), dementia and chronic kidney disease (10 each one). Most of the CPs on rare diseases have been edited by Regions with an established Rare Disease Network, i.e., Lombardia (125 CPs), Lazio (74) and Toscana (40): neurology (61) and oncology (52) were the most represented clinical areas. CONCLUSIONS: The high number of CPs approved in Italy confirms an increasing interest of the healthcare institutions. The collected CPs show an extreme variety of titles, text structures and disease choices. Given the absence of an institutional observatory and of devotees of shared and harmonized CPs, annually Pdta Net makes available an updated and complete overview of these governance tools, which are essential for the upcoming changes of the Italian national health service.

[Community acquired pneumoniae and chronic obstructive pulmonary disease: the RWE analysis of events that required hospitalizations, and healthcare integrated costs.] / Polmonite acquisita in comunità e broncopneumopatia cronica-ostruttiva: analisi RWE degli eventi che hanno richiesto il ricovero ospedaliero e dei costi assistenziali associati.

INTRODUCTION: The community-acquired pneumonia (Cap) and the acute exacerbation of chronic obstructive pulmonary disease (Aecopd) frequently receive wrong therapies, leading to the increase of healthcare consumption resources, direct and indirect costs, and antimicrobial resistance. This study identified Cap and Aecopd hospitalized events, and analyzed them in terms of comorbidities, antibiotic use, re-hospitalizations, diagnostics and costs, from the perspective of the Italian national health service (Inhs). METHODS: From the database of Fondazione Ricerca e Salute (ReS), hospitalizations for Cap and Aecopd from 2016 to 2019. Demographics, comorbidities and mean in-hospital stay at the baseline, antibiotics reimbursed by the Inhs within 15 days before and after the index event, outpatient diagnostics performed before the event and in-hospital diagnostics, and direct costs charged to the Inhs, are assessed. RESULTS: From 2016 to 2019 (~5 million inhabitants/year), 31,355 events of Cap (1.7x1000/year) and 42,489 events of Aecopd (4.3x1000 inhabitants aged ≥45/year) were identified, of which 32% and 26.5%, respectively, were treated with antibiotics before the hospitalization. The highest frequency of hospitalizations and comorbidities, and the longest mean in-hospital stays are found among elderly. Events not treated before and after the hospitalization showed the longest in-hospital stay. More than 12 Ddd (defined daily dose) are dispensed after the discharge. Local outpatient diagnostics are performed before the admission to <1% of the events; in-hospital diagnostics are registered in 5.6% and 1.2% of Cap and Aecopd, respectively, discharge forms. About 8% and 24% of Cap and Aecopd, respectively, are re-hospitalized during one subsequent year, mainly within one month. The mean expenditures per event of Cap and Aecopd were € 3646 and € 4424, respectively: hospitalizations, antibiotics and diagnostics accounted for the 99%, 1% and <0.1% of the total expense, respectively. CONCLUSIONS: This study provided a very high dispensation of antibiotics after the hospitalization for Cap and Aecopd, while a very low use of differential diagnostics available within the observed periods, to the detriment of the enforcement actions proposed at institutional levels.

[Occurrence of cardiovascular events within a large population of patients affected by rheumatoid arthritis with or without diabetes.] / Sviluppo di eventi cardiovascolari in un'ampia popolazione di pazienti affetti da artrite reumatoide con o senza diabete.

INTRODUCTION: Rheumatoid arthritis (Ra) and diabetes are often associated with chronic multimorbidity and share the high risk of development of major cardiovascular events (Mace). This study aimed to identify and analyse patients with only Ra, Ra + diabetes, and only diabetes, in terms of comorbidities and new occurrence of Cv events, from the perspective of the Italian national health service (Inhs). METHODS: Starting from the Fondazione ricerca e salute (ReS)'s database, through the record linkage of administrative healthcare data, cohorts with only Ra, Ra + diabetes and only diabetes have been selected, characterized (age and sex), and analysed by comorbidity (depression, dyslipidemia, hypertension, hemorrhagic stroke and ischemic stroke/transient ischemic attack - Tia, coronary artery disease - Cad, heart failure - Hf, chronic liver disease, periphery artery disease - Pad, chronic kidney disease, asthma/chronic obstructive pulmonary disease - Copd, neoplasia) and by new Cv events (Hf, Cad and ischemic stroke/Tia) within two follow-up years (Kaplan-Meier curves). A logistic regression model defined contribution and type of association of some variables on new Cv events. RESULTS: In 2018, from 5.375.531 Inhs beneficiaries in the ReS database, 13.698 (0.25%) were affected by only Ra, 1728 (0.03%) by Ra + diabetes, 347,659 (6.8%) by only diabetes. The only Ra cohort was composed by more females, younger and with less comorbidities patients. Proportions of 79.3%, 70.8% and 38.5% of patients with Ra + diabetes, only diabetes and only Ra were affected by 2 to ≥4 comorbidities: among patients with Ra + diabetes, comorbidities showed the highest frequencies, mainly hypertension, dyslipidemia and asthma/Copd. Within two follow-up years, about 8% of patients with diabetes with/without Ra developed a new Cv event (vs 3% with only Ra). The presence of Ra/diabetes or Ra + diabetes, male sex, older age and comorbidities of interest resulted significantly (p<0.01) associated with a higher Cv risk. CONCLUSIONS: Comorbidities and the co-presence of diabetes in patients with Ra determine a complicated framework with high risk of Cv events. It is worthy include more complex patients in clinical trials, in order to generate evidence useful for even more multidisciplinary medical teams.

[The population affected by neovascular age-related macular degeneration and treated with anti-Vegf through Italian administrative healthcare data.] / La popolazione con degenerazione maculare neovascolare correlata all'età trattata con anti-Vegf attraverso i dati amministrativi sanitari.

INTRODUCTION: The neo-vascular age-related macular degeneration (nAmd) is a frequent cause of vision loss, although the intravitreal (Ivt) injections of anti-Vegf (vascular endothelial growth factor) have improved functional outcomes. This study has assessed the healthcare and economic burden on the Italian national health service (Inhs) for patients with nAmd and new users of anti-Vegf. METHODS: From the database of Fondazione Ricerca e Salute (ReS), people aged ≥55 and with an in-hospital diagnosis of nAmd and/or an injection of anti-Vegf (aflibercept, ranibizumab, pegaptanib; index date) in 2018 are selected. Those with other conditions treated with anti-Vegf and with an Ivt injection before 2018 are excluded. New users of anti-Vegf are analyzed by sex, age, comorbidities, Ivt administrations, switch of anti-Vegf, local outpatient specialist services (with some focuses) and direct healthcare costs charged to the Inhs Results. In 2018, of 8125 inhabitants aged ≥55 with nAmd (4.6x1000 inhab.; mean age 76±9; F: 50%), 1513 (19%) are new users of Ivt anti-Vegf (mean age 74±9), whose incidence (0.9x1000) increased with age until 84 years old. A proportion of 60.7% had ≥2 comorbidities (mainly hypertension, dyslipidemia and diabetes). Within the 2nd follow-up year, only 598 patients are still treated (60% were lost). On average, 4.8 Ivt injections in the first and 3.1 in the second year are registered. On average, the total cost charged to the Inhs per new user of anti-Vegf was € 6726 (Ivt anti-Vegf accounted for the 76%) and € 3282 (hospitalizations for causes different from nAmd accounted for the 47%), during the first and the second year, respectively. CONCLUSIONS: This analysis suggests that in Italy people with nAmd and new users of anti-Vegf are elderly, affected by many comorbidities, treated with Ivt anti-VEGF less than what is required and authorized to achieve a benefit, undergo very few follow-up outpatient specialist visits and tests and, within the 2nd year, their hospitalizations for causes different from nAmd mainly weighs on the total expenditure charged to the Inhs.

A Drug Screening Revealed Novel Potential Agents against Malignant Pleural Mesothelioma.

The lack of effective therapies remains one of the main challenges for malignant pleural mesothelioma (MPM). In this perspective, drug repositioning could accelerate the identification of novel treatments. We screened 1170 FDA-approved drugs on a SV40-immortalized mesothelial (MeT-5A) and five MPM (Mero-14, Mero-25, IST-Mes2, NCI-H28 and MSTO-211H) cell lines. Biological assays were carried out for 41 drugs, showing the highest cytotoxicity and for whom there were a complete lack of published literature in MPM. Cytotoxicity and caspase activation were evaluated with commercially available kits and cell proliferation was assayed using MTT assay and by clonogenic activity with standard protocols. Moreover, the five most effective drugs were further evaluated on patient-derived primary MPM cell lines. The most active molecules were cephalomannine, ouabain, alexidine, thonzonium bromide, and emetine. Except for alexidine, these drugs inhibited the clonogenic ability and caspase activation in all cancer lines tested. The proliferation was inhibited also on an extended panel of cell lines, including primary MPM cells. Thus, we suggest that cephalomannine, ouabain, thonzonium bromide, and emetine could represent novel candidates to be repurposed for improving the arsenal of therapeutic weapons in the fight against MPM.

Pro64His (rs4644) Polymorphism Within Galectin-3 Is a Risk Factor of Differentiated Thyroid Carcinoma and Affects the Transcriptome of Thyrocytes Engineered via CRISPR/Cas9 System.

Background: Galectin-3 (LGALS3) is an important glycoprotein involved in the malignant transformation of thyrocytes acting in the extracellular matrix, cytoplasm, and nucleus where it regulates TTF-1 and TCF4 transcription factors. Within LGALS3 gene, a common single-nucleotide polymorphism (SNP) (c.191C>A, p.Pro64His; rs4644) encoding for the variant Proline to Histidine at codon 64 has been extensively studied. However, data on rs4644 in the context of thyroid cancer are lacking. Thus, the aim of the present work was to evaluate the role of the rs4644 SNP as risk factor for differentiated thyroid cancer (DTC) and to determine the effect on the transcriptome in thyrocytes. Methods: A case/control association study in 1223 controls and 1142 unrelated consecutive DTC patients was carried out to evaluate the association between rs4644-P64H and the risk of DTC. We used the nonmalignant cell line Nthy-Ori (rs4644-C/A) and the CRISPR/Cas9 technique to generate isogenic cells carrying either the rs4644-A/A or rs4644-C/C homozygosis. Then, the transcriptome of the derivative and unmodified parental cells was analyzed by RNA-seq. Genes differentially expressed were validated by quantitative reverse transcription PCR and further tested in the parental Nthy-Ori cells after LGALS3 gene silencing, to investigate whether the expression of target genes was dependent on galectin-3 levels. Results: rs4644 AA genotype was associated with a reduced risk of DTC (compared with CC, ORadj = 0.66; 95% confidence interval = 0.46-0.93; Pass = 0.02). We found that rs4644 affects galectin-3 as a transcriptional coregulator. Among 34 genes affected by rs4644, HES1, HSPA6, SPC24, and NHS were of particular interest since their expression was rs4644-dependent (CC>AA for the first and AA>CC for the others), also in 574 thyroid tissues of Genotype-Tissue Expression (GTEx) biobank. Moreover, the expression of these genes was regulated by LGALS3-silencing. Using the proximity ligation assay in Nthy-Ori cells, we found that the TTF-1 interaction was genotype dependent. Conclusions: Our data show that in thyroid, rs4644 is a trans-expression quantitative trait locus that can modify the transcriptional expression of downstream genes, through the modulation of TTF-1.

Variation rs2235503 C > A Within the Promoter of MSLN Affects Transcriptional Rate of Mesothelin and Plasmatic Levels of the Soluble Mesothelin-Related Peptide.

Soluble mesothelin-related peptide (SMRP) is a promising biomarker for malignant pleural mesothelioma (MPM), but several confounding factors can reduce SMRP-based test's accuracy. The identification of these confounders could improve the diagnostic performance of SMRP. In this study, we evaluated the sequence of 1,000 base pairs encompassing the minimal promoter region of the MSLN gene to identify expression quantitative trait loci (eQTL) that can affect SMRP. We assessed the association between four MSLN promoter variants and SMRP levels in a cohort of 72 MPM and 677 non-MPM subjects, and we carried out in vitro assays to investigate their functional role. Our results show that rs2235503 is an eQTL for MSLN associated with increased levels of SMRP in non-MPM subjects. Furthermore, we show that this polymorphic site affects the accuracy of SMRP, highlighting the importance of evaluating the individual's genetic background and giving novel insights to refine SMRP specificity as a diagnostic biomarker.

Progress of malignant mesothelioma research in basic science: A review of the 14th international conference of the international mesothelioma interest group (iMig2018).

Here we summarize the most recent update of mesothelioma research in basic science presented at the 14th iMig2018 international conference. The symposium of basic science track mainly focused on the drivers of mesothelioma initiation and progression, molecular pathogenesis, and perspectives on potential therapeutic approaches. This review covers several promising fields including strategies efficiently inhibiting YAP/TAZ functions or their critical downstream targets, heparanase inhibitors, RAN depletion, and MIF/CD74 inhibitors that may be developed as novel therapeutic approaches. In addition, targeting mesothelioma stem cells by depleting M2-polarized macrophages in tumor microenvironment or blocking Tnfsf18 (GITRL)-GITR signalling might be translated into therapeutic modalities in mesothelioma treatment.